Role of Akt in proliferation mechanism dependent on redox state.

ANTICO ARCIUCH VG; GALLI S; FRANCO MC; CARRERAS MC; PODEROSO JJ

Aguas de Lindoia, Brasil

Congreso; IV Meeting of the South American Group of the Society for Free Radical Biology and Medicine; 2005

South American Group of the Society for Free Radical Biology and Medicine

Akt is an important regulator of cellular processes such as proliferation and apoptosis, angiogenesis and glucose metabolism. Akt becomes activated by different stimuli that include growth factors, cytokines, steroid hormones and cellular stress. The aim of this work was to study H2O2 modulation of Akt traffic in connection with cell cycle progression of cell line NIH/3T3. Subcellular distribution of P-Akt (nuclei, mitochondria and cytosol) was followed by western blot. Akt activity was detected by an in vitro kinase assay using GSK-3 fusion protein as a substrate. Proliferation and apoptosis were evaluated by [H3]thymidine incorporation, flow cytometry with propidium iodide staining and acridine orange/ethidium bromide staining. Mitochondrial Akt localization was assessed by confocal microscopy with Mitotracker Red. In NIH/3T3 cells, 100 µM H2O2 promoted proliferation, and > 250 mM H2O2 apoptosis, with differential set up of Akt pathways. At 100 µM H2O2, Akt activation and translocation to nucleus were rapid and persistent while 250 µM H2O2 induced transient activation. 100 ìM H2O2 promoted a rapid activation of Akt in cytosol while 250 µM H2O2 did not. Interestingly, 100 µM H2O2 did not seem to elicit modulation of mitochondrial Akt while 250 ìM H2O2 induced the entrance of Akt to the organelle. In addition, Akt activity decreased after 15 minutes of 100 µM H2O2 incubation in this fraction. The results suggest that H2O2 differentially activates Akt, and that this activation and the traffic to mitochondria regulate cell cycle progression.